The present invention relates to a method of modifying a polymer surface by covalent attachment of functional compounds, also designated ligands.
1. The Technical Field
Products made of synthetic or natural polymers having modified surfaces are very important in many technical areas.
Surface modification of polymers by the introduction of various functional groups or the covalent attachment of biologically active molecules has been the subject of increased research in recent years in such different areas as the development of novel biocompatible implants, for biosensors and biomaterials, for affinity chromatography, for surface resistant materials, for biosensors, and for covalent immobilization of high or low molecular weight molecules in ELISA assays.
2. Prior Art
Thermochemical Methods
Most methods involve sequential treatment of the polymer surface with chemical reagents to introduce functional groups to function as handles for coupling of a functional compound also called ligand. However, these methods usually employ hazardous chemicals and several time-consuming steps. In addition to this, only a limited number of methods are described in which the mechanical and optical properties of the polymer can be preserved. A method of introducing primary amino groups onto polystyrene tubes using thermochemical reactions and onto microtitre plates has been described by Alexio, J. A. G.; Swaminathan, B; Minnich, S. A.; Wallshein, V. A.; J. Immunoassay 1985, 6, 391-407.
Radioanalytical Methods
EP-A-O 155 252 discloses a method of preparing an immunoreactive solid phase wherein a biologically active molecule is covalently bound to functional groups of vinyl monomers radiation grafted to a solid polymer surface. Grafting requires an adequate radiation dose under an inert atmosphere using radiation such as ultraviolet or ionizing radiation. Specific examples using 0,25 Mrad/h .sup.60 Co irradiation source for 10-12 hours are given.
International application no. WO 91/02768 discloses radio-derivatized polymers produced by contacting non-polymerizable conjugands, such as quinones or compounds from which quinones or quinoid structures are generated during radio-derivatization, with radiolyzable polymers, such as polystyrene, in the presence of high energy gamma rays. The radio-derivatized polymers are suitable for introducing anchoring groups for covalent immobilization or for fixing of molecules on polymer surfaces with or without cross-linkers or with activators such as carbodiimides.
A disadvantage of radio-derivatization is the use of ionizing high energy gamma rays which requires costly health physical precautions in carrying out the method.
Photochemical Methods
A number of photochemical methods of modifying polymer surfaces are also known. In these methods a desired ligand (L)--often a sensitive biomolecule--is immobilized on the polymeric material surface (P) through a photochemically reactive group (Q) and a spacer (S) and optionally a thermochemical reactive group (T).
In general, the covalent attachment of the desired molecule (L) to the surface can be established in three ways:
1) The photochemically reactive group (Q) which is coupled--via a spacer (S)--to a thermochemical reactive group (Q-S-T) is bound covalently to the surface (P) by a photochemical reaction (P-Q-S-T). Subsequently, the desired molecule (L) is coupled to the surface (P-Q-S-T) by thermochemical reaction (P-Q-S-T-L).
2) The photochemically reactive group (Q) which is coupled directly--via a spacer (S)--to the desired molecule (Q-S-L) is bound to the surface (P) by a photochemical reaction (P-Q-S-L).
3) The photochemically reactive group (Q) is coupled covalently to the surface (P) by a thermochemical reaction (P-Q). Subsequently, the desired molecule (L) is coupled to the surface (P-Q) by a photochemical reaction (P-Q-L).
The first two strategies are potentially the most flexible ones and allow control of the orientation of the immobilized ligand.
EP-A2-0 319 953 discloses a photochemical method of modifying a polymer surface by immobilizing an optionally substituted two or three membered heterocyclic compound to the surface of the polymer using electromagnetic irradiation with a wavelength shorter than 700 nm. Preferred compounds are optionally substituted coumarins, benzofurans, indols, and angelicins. Particularly, optimally substituted psoralens are preferred.
A disadvantage of this method is that psoralens are multifunctional compounds which are not easy to synthesize. They are expensive and not chemically stable, e.g. spacers containing primary amines (as a functional group) can not be introduced onto the surface, because the amine will react with the psoralen.
When irradiated with UV light having a short wavelength, a secondary amine placed in the end position and coupled--via a spacer--to psoralen can be photochemically bound to a polystyrene surface. When biotin is coupled to the spacer derivative, biotin can also be photochemically bound to polystyrene surfaces and polymethyl-methacrylate particles. The method cannot be considered to be generally applicable, as only these two examples work satisfactorily. The photochemical mechanism has not been fully understood, but it is known that psoralen derivatives react with double bonds in a 2+2 cyclo addition reaction when irradiated with UV light.
A number of patent publications U.S. Pat. Nos. 4,722,906, 4,973,493, 5,002,582 and PCT/US88/04491, assigned to Biometric Systems Inc., disclose methods for photo-chemical modification of polymer surfaces. The patent publications essentially describe methods involving activating latent reactive groups selected from the group consisting of those able to generate free radicals, carbenes, nitrenes and exited states of ketones, and covalently bonding thereof to a solid surface.
The disclosed latent reactive groups responsive to ultra-violet, visible or infrared portions of the electromagnetic spectrum are: azides, acylazides, azido formates, sulfonyl azides, phosphoryl azides; diazo compounds such as diazoalkanes, diazoketones, diazoacetates, beta-ketone-alpha-diazoacetates; aliphatic azo compounds, diazirines, ketone, diphenylketone and photoactivable ketones such as benzophenone and acetophenone; and peroxy compounds such as dialkyl- and diacyl peroxides and peroxyesters.
Latent reactive groups, which upon irradiation with high energy UV light generates highly reactive radicals, carbenes or nitrenes, suffer from a number of drawbacks. Such species are extremely reactive and will either rear-range or immediately react with most organic compounds, organic solvents and water. When the irradiation takes place in a solution, this results in loss of photoreagent and ineffecient or reaction with the polymer surface. The simple precursors requires long irradiation times (typically 12 hours) which makes the application of these as photoreactive groups time consuming, inefficient and not suitable for immobilization of sensitive biomolecules.
Nothing is indicated nor suggested about photochemical coupling using quinones as the photoreactive group.